This application claims Paris Convention priority of DE 199 49 953.5 filed Oct. 16, 1999 the complete disclosure of which is hereby incorporated by reference.
The invention concerns a total reflection measuring cell for the spectroscopic examination of an, in particular, fluid sample in an infrared (=IR) spectrometer comprising an ATR (=attenuated total reflectance) crystal having a plane surface and a diaphragm clamped on several sides in a clamping frame, wherein the clamping frame is disposed such that the diaphragm extends at a small separation from the plane surface of the ATR crystal.
ATR measuring cells of this type are known from U.S. Pat. No. 5,362,445 A1 or DE 196 12 877 C1.
To study the interactions between biomolecules by means of FTIR-ATR spectroscopy, it is important to introduce ligands in the evanescent field without causing mechanical disturbances of the sensitive layer on the ATR crystal or changing the concentrations of the dissolved substances which interact with the immobilized molecules. This is realized by a diaphragm, generally a dialysis diaphragm which is disposed above the sensitive crystal surface and separates a sample chamber below the diaphragm from a dialysis chamber above the diaphragm. Compounds having a low molecular weight can be dialyzed into and out of the sample chamber according to the MW Cut-Off of the diaphragm without lessening macromolecules in the evanescent field or destroying the immobilized thin layers on the crystal surfaces. Introduction of the ligands into the evanescent field can be accelerated beyond the thermal diffusion rate. Through the use of electrophoretical currents, charged ligands can be specifically transported into and out of the sample chamber through the diffusion barrier and the mechanical obstacle formed by the dialysis diaphragm.
The construction and use of such total reflection measuring cells through the initially mentioned features is described in detail in the above cited DE 196 12 877 C1, the complete disclosure of which is hereby incorporated by reference. The use of a diaphragm in an ATR cell includes, however, the problem of keeping an exact distance from the plane surface of the ATR crystal. Unfortunately, the diaphragms clamped in a clamping frame have generally the tendency to form waves or sag through. The filling or emptying of the hollow space above the diaphragm can cause pressure transfer to the hollow space below the diaphragm such that the sensitive sample layer on the crystal is damaged and the IR measurement becomes impossible. In the extreme case, the diaphragm will sag that much that during filling/removing processes it comes even in direct contact to the crystal surface thus removing the immobilized sample from the crystal.
It is therefore the object of the present invention to present a total reflection measuring cell of the initially mentioned type which does not have the above-mentioned disadvantages, guarantees a considerably increased measuring accuracy and thus reliably ensures the quality of the measuring results.
In accordance with the invention, this is achieved by additionally providing a tensioning device for displacing the diaphragm on an inner partial area of its surface clamped between the legs of the clamping frame in the direction of the plane surface of the ATR crystal such that the displaced inner partial area of the diaphragm surface has a higher tension than before the diaphragm surface merely clamped in the clamping frame, and providing a stop to which the tensioning device is forced and fixed in this position such that the inner partial area of the diaphragm surface has an identical defined distance from the plane surface of the ATR crystal.
The tensioning device reliably prevents sagging of the diaphragm in the tensioned inner partial area such that the natural waviness of the diaphragm material does not present any problems any more. The mechanical stop achieves an extremely exact positioning of the tensioned diaphragm surface in the inner partial area relative to the plane surface of the ATR crystal, in particular an exact parallelism with simple means and without great production effort.
Particularly preferred is an embodiment of the inventive ATR measuring cell wherein the tensioning device comprises a continuous wedge whose continuous closed edge line tensions the inner partial area of the diaphragm and forces same towards the ATR crystal. A continuous wedge of this type can be produced in a mechanically simple manner and with high accuracy.
In an advantageous further development of this embodiment, the angle of the wedge sides of the continuous wedge towards the plane surface of the ATR crystal in the fixed position of the tensioning device is between 30xc2x0 and 60xc2x0, preferably approximately 45xc2x0. In general, one will choose a symmetrical wedge shape, i.e. both wedge sides having the same angle. Variants are also feasible wherein the two wedge sides have different angles.
Due to the longitudinal shape of the common ATR crystals, a further development is preferred wherein the continuous enclosed edge line of the continuous wedge forms a longitudinal oval comprising two parallel longitudinal sides forming a semi-circle at each of their ends.
One embodiment is particularly preferred, wherein the stop towards which the tensioning device can be forced and fixed in this position comprises a continuous inclined surface of an inclination corresponding to the wedge angle. This guarantees positioning of the diaphragm tensioned in the inner partial area plane-parallel to the sensitive surface of the ATR crystal with high precision whereby the production effort is very small.
A further advantageous embodiment of the inventive ATR measuring cell is characterized in that between the inner partial area of the tensioned diaphragm surface within the continuous enclosed edge line of the continuous wedge and a cover part of the tensioning device facing away from the diaphragm in the unfolded state, a hollow volume is formed which can be filled or emptied via an inlet and an outlet in the cover part. The sample fluid can flow through this hollow space when fluid samples are measured.
A further development is advantageous, wherein the cover part is produced from transparent material, preferably plexiglass. This allows in particular observation of the flow behaviour of the sample fluid but also discovery of possible contamination in the hollow space and removal thereof through rinsing.
Also preferred is one embodiment wherein a hollow volume is formed between the plane surface of the ATR crystal and the tensioned inner partial area of the diaphragm surface which can be filled or emptied via an inlet and an outlet. In this manner, the molecules diffused through the diaphragm can contact the sensitive surface of the ATR crystal. Moreover, the inlet and outlet allow rinsing of the sensitive area of the ATR crystal for cleaning which may serve i.a. to prepare subsequent measurements.
One embodiment of the inventive ATR cell is particularly preferred wherein the tensioning device can be detachably and rigidly connected, preferably screwed to a base plate onto which the ATR crystal is fixed. This considerably facilitates the relative positioning and fixing of the achieved optimum position between diaphragm and ATR crystal.
One further development of this embodiment is particularly preferred for geometrical reasons, wherein the inlet and outlet lead, through the base plate, to the hollow space between the plane surface of the ATR crystal and the tensioned inner partial area of the diaphragm surface.
In a further development, the optimum relative position can be achieved and fixed in that the clamping frame comprising the clamped diaphragm is disposed between the base plate and the tensioning device and comprises through-bores for receiving and guiding the screws for screwing the tensioning device to the base plate.
In a particularly simple further development of these embodiments, the stop for the tensioning device is integrated in the base plate.
One further development is particularly advantageous with respect to handling of the respective individual parts of the inventive ATR measuring cell, wherein the continuous inclined surface of the stop is disposed on the base plate side facing the diaphragm in the mounted state and surrounds an inside width through the base plate wherein on the other side of the base plate, the ATR crystal is fixed with its plane surface facing the inside width.
The diaphragm used in the inventive ATR measuring cell is usually formed as dialysis diaphragm. In embodiments, the ATR measuring cell itself can be formed as dialysis cell.
Alternatively, the ATR measuring cell can also be formed as electrophoresis cell.
To allow a simple possibility for both measuring methods, the inventive ATR measuring cell can be constructed such that two exchangeable cover parts can be mounted onto a fixed base plate part comprising the ATR crystal fixed thereon or thereto, wherein the cover parts contain the respectively used diaphragms with the inventive tensioning devices and are especially designed either for dialysis or electrophoresis measurements. Such an ATR measuring cell meets the most important requirements for infrared studies on a large plurality of macromolecular interactions. The unit generally contains a trapezoidal internal reflection element in the form of the ATR crystal which is disposed in a rinsable casing with regulated temperature and completely encapsulated. The base plate onto which the ATR crystal is fixed is generally advantageously coated with teflon.
In an advantageous embodiment of the inventive ATR measuring cell, a further clamping frame with clamped diaphragm and a further tensioning device are disposed on the side of the first diaphragm facing away from the ATR crystal in the mounted state, parallel and at a defined separation from same. This embodiment is suited in particular for electrophoresis examinations.
A further development of this embodiment is characterized in that both tensioning devices comprise two continuous wedges arranged next to one another and extending parallel to the ray guidance axis of the ATR crystal. Their continuous enclosed edge lines tension the respective inner partial area of the diaphragms disposed in parallel and force same towards the ATR crystal wherein the tensioning device located closer to the ATR crystal comprises on its side facing away from the ATR crystal a stop for the tensioning device further away from the ATR crystal which comprises two continuous inclined surfaces whose inclination corresponds with the wedge angle of the two continuous edge lines of the tensioning device further away from the ATR crystal.
This further development can be improved in that the two tensioning devices disposed on top of one another are formed such that when mutual fixing, the diaphragm tensioned between them is forced between the two continuous wedges of the tensioning device further away from the ATR crystal such that it represents a large Ohmic resistance in this area. This prevents undesired xe2x80x9ccross flowsxe2x80x9d via the tensioned diaphragm.
One further development is also preferred wherein the tensioning device less remote from the ATR crystal comprises recesses within the two continuous wedges such that two hollow spaces are generated between the two unfolded diaphragms which can be separately filled via a separate inlet and a separate outlet within the tensioning device closer to the ATR crystal.
Moreover, it is favourable to generate a hollow space between the unfolded diaphragm less remote from the ATR crystal and the ATR crystal which can be filled and emptied via an inlet and an outlet within the base plate to which at least one of the two tensioning devices is mounted.
Finally, a further improvement can be achieved in that the tensioning device further away from the ATR crystal comprises recesses within the two continuous wedges which are connected in a direction opposite to the two tensioned diaphragms separately with two separate hollow spaces such that after filling all hollow spaces of the mounted ATR cell with an electrically conducting liquid and after applying an electric voltage to the hollow spaces, which are connected with the recesses of the tensioning device further away from the ATR crystal, a current flux can be generated which flows from the hollow space connected with a cathode to the hollow space connected with an anode passing both diaphragms twice.
Further advantages of the invention can be gathered from the description and the drawing. The features mentioned above and below can be used in accordance with the invention either individually or collectively in any arbitrary combination. The embodiments shown and described are not to be understood as exhaustive enumeration but have exemplary character for describing the invention.
The invention is shown in the drawing and is explained in more detail with reference to an embodiment: